紫花苜蓿Ⅲ型几丁质酶基因同源克隆与序列变异分析

Homologous Cloning and Sequence Variation Analysis of TypeⅢ Chitinase Gene in Alfalfa

型几丁质酶(chitinaseⅢ, ChiⅢ)是植物重要的防卫蛋白之一,可阻止病原菌的侵入和发展,抑制特定病害的发生,在植物抵抗病原菌的过程中具有重要作用。为了分离苜蓿ChiⅢ基因的基因组序列,进而分析其序列变异情况,本研究采用同源克隆法,从16个紫花苜蓿(Medicago sativa)品种的集群DNA中分离了9个属于a类的ChiⅢ基因(ChiⅢa)的部分基因组编码序列,分别与蒺藜苜蓿(Medicago truncatula)的9个ChiⅢa基因相对应,将其命名为chit3-1-1、chit3-1-2、chit3-1-4、chit3-1-5、chit3-1-6、chit3-1-9、chit3-1-10、chit3-1-11和chit3-1-12。序列变异分析显示:chit3-1-1、chit3-1-2、chit3-1-5、chit3-1-9、chit3-1-10、chit3-1-11和chit3-1-12这7个基因的单核苷酸多态性(SNP)位点间的平均距离分别为28 bp、27 bp、648 bp、55 bp、64 bp、130 bp和131 bp,差异较大。表明chit3-1-5序列高度保守,chit3-1-11和chit3-1-12比较保守,chit3-1-9和chit3-1-10变异中等,而chit3-1-1和chit3-1-2的变异较大。从总体看,在苜蓿基因组中ChiⅢa基因的序列较为保守。本试验为进一步对ChiⅢ基因进行关联分析等遗传学研究和cDNA全长克隆等分子生物学研究提供了良好的基础。

ChitinaseⅢ(ChiⅢ) is one of the most important defensive proteins in plant.Because they could be able to prevent from intrusion of pathogenic fungi,slower developing of diseases,and finally restrain occurring of diseases,thus they play an important role in plant resistance to pathogens.This study aimed to isolate the genomic coding sequences of ChiⅢ genes,and then analyze their sequence variations by a homology-based cloning method using the bulked DNA from 16 cultivars as materials in alfalfa(Medicago sativa).Consequently,the part genomic coding sequences of a total of 9 ChiⅢ genes were isolated,and all of them belonged to the type a(ChiⅢa).Each of these 9 alfalfa ChiⅢa genes had a corresponding homologous ChiⅢa gene from Medicago truncatula,and they were named as chit3-1-1,chit3-1-2,chit3-1-4,chit3-1-5,chit3-1-6,chit3-1-9,chit3-1-10,chit3-1-11 and chit3-1-12.Sequence vibration analysis showed that among the found 9 alfalfa Chi Ⅲa genes,a total of 7 genes,including chit3-1-1,chit3-1-2,chit3-1-5,chit3-1-9,chit3-1-10,chit3-1-11 and chit3-1-12,possessed a divergent average inter-SNP distance with a value of 28 bp,27 bp,648 bp,55 bp,64 bp,130 bp and 131 bp,respectively.This indicated chit3-1-5 was highly conserved while chit3-1-1 and chit3-1-2 displayed a highly variable sequence variation.Meanwhile,chit3-1-9 and chit3-1-10 had a median sequence variation and chit3-1-11 and chit3-1-12 were slightly conserved.On the whole,the ChiⅢa genes found were relatively conservative in alfalfa genome.This experiment provides a good basis for further genetic research such as association analysis of Chi Ⅲgenes and molecular biology research such as full-length cloning of cDNA.The experiment provides a good basis for further genetic research such as the association analysis of Chi Ⅲ gene and molecular biology study such as full-length cloning of cDNA.